This proposal outlines experiments to determine the mechanisms by which the envelope glycoprotein of vesicular stomatitis virus is inserted into the host cell endoplasmic reticulum membrane as well as how intracellular transport of the glycoprotein through the Golgi complex to the plasma membrane is achieved. This study of the steps in viral infection will help elucidate the mechanisms ordinarily used by the host cell in membrane assembly. The biochemical steps in the insertion process will be dissected by the purification of membrane proteins from pancreatic microsomes that are required for insertion of the viral glycoprotein into these membranes in a cell-free protein synthesis system which has already been developed. This in vitro system will also permit a clear test of predictions made from the "signal hypothesis", developed recently to explain key aspects in the initiation of protein secretion across membranes. The approach to the problem of intracellular transport will first involve the identification of the vesicles which transport the viral glycoprotein from the endoplasmic reticulum to the Golgi complex. Available evidence encourages the belief that small coated vesicles provide this transport function. This notion will be rigorously tested with pulse-chase experiments using viral-infected cells, from which pure preparations of coated vesicles can be obtained. The possibility that coated vesicle formation and "intracellular" transport can take place in cell lysates, as inferred from recently published findings, will also be investigated.